Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis

A unique nanoporous sol-gel glass possessing a highly ordered porous struct ure (with a pore size of 153 Angstrom in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, a-chymotrypsi n, was efficiently bound onto the glass via a bifunctional ligand, trimetho xysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha -chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either i n aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme ina ctivation caused by structural denaturation. In addition, the activity of t he immobilized alpha -chymotrypsin was also much higher than that of the na tive enzyme in various organic solvents. From these results, it appears tha t the glass-enzyme complex developed in the present work can be used as a h igh-performance biocatalyst for various chemical processing applications, p articularly in organic media.